A vast majority of biologically active molecules including growth factors, cytokines, neurotransmitters and hormones transduce signals via specific cell-surface receptors. Some of these receptors are then coupled to heterotrimeric GTP-binding proteins (G proteins) which, upon activation, relay signals to a variety of cellular effectors including at least four phospholipase C (PLC) variants and adenylyl cyclases.
G proteins mediate external signals by forming heterotrimers consisting of an alpha, beta and gamma subunit. Several isoforms of each subunit have been identified and therefore, through subunit heterogeneity, G proteins effectively integrate multiple signaling cascades. The alpha subunits of G proteins contain the GTP binding site and intrinsic catalytic GTPase activity. Based on sequence similarity and function, these subunits have been classified into four major groups; Gs, which stimulate adenylyl cyclases; Gi, which inhibit adenylyl cyclases; Gq, which activate PLC isoforms and G12/13, which mediate pathways associated with cell growth and differentiation (Hamm, J. Biol. Chem., 1998, 273, 669-672).
G-alpha-i3 is a member of the Gi subfamily of G proteins which is involved in hormonal inhibition of adenylyl cyclase and in the regulation of plasma membrane enzymes. G-alpha-i3 has also been shown to mediate dopamine, thyrotropin-releasing hormone and somatostatin signal transduction pathways (Kineman et al., Endocrinology, 1994, 135, 790-793; Kineman et al., J. Endocrinol., 1996, 148, 447-455; Law et al., J. Biol. Chem., 1993, 268, 10721-10727). In addition, an upstream inhibitor of phospholipase C, U73122, resulted in the inhibition of Gi-mediated protein activation further implicating G-alpha-i3 in lipid signaling cascades (Wu et al., Neuroreport., 1998, 9, 99-103).
Comparison studies of modified forms of the pertussis toxin-insensitive form of G-alpha-i3 (pertussis toxin is normally an inhibitor of Gi function) and the wild type protein, in assays designed to investigate the interactions of adrenoceptors and Gi proteins, demonstrated that more agonist was required to stimulate the mutant protein than the wild type. These studies showed that the affinity of the wild type Gi protein for the receptor was greater than that of the mutant (Wise et al., Biochem. J., 1997, 321, 721-728).
In human hepatocellular carcinoma (HCC), the expression and functional activity of G-alpha-i3 was increased in 80% of the tumors examined. These results indicate that the regulation of the adenylate cyclase system in these cells may contribute to the formation or progression of the carcinoma (Schmidt et al., Hepatology, 1997, 26, 1189-1194).
The expression of G-alpha-i3 has also been shown to be regulated by physiologic shear stresses such as flow. Using a transcapillary coculture system, it was shown that G-alpha-i3 expression was increased by high-flow conditions in endothelial and vascular smooth muscle cells implicating Gi proteins in flow-induced responses of vessel wall function (Redmond et al., Arterioscler. Thromb. Vasc. Biol., 1998, 18, 75-83).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of G-alpha-i3 and to date strategies aimed at inhibiting or investigating G-alpha-i3 function have predominantly involved the use of antibodies.
These strategies, however, are untested as therapeutic protocols. Consequently there remains a long felt need for additional agents capable of effectively inhibiting G-alpha-i3 function and antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of G-alpha-i3 expression.